The invention relates generally to mass spectrometer (MS) instruments and specifically to mass spectrometers which utilize a matrix assisted laser desorption ionization (MALDI) ion source. More specifically, the invention relates to MALDI sources that are operated at an elevated pressure of from about 0.1 torr to about 10 torr, in order to assist in the MS analysis of labile molecules, such as proteins and peptides.
The MALDI method, an established technique for analysis of biopolymers (see, e.g., M. Karas, D. Bachmann, U. Bahr and F. Hillenkamp, Int. J. Mass Spectrom Ion Processes 78 (1987), 53; Anal. Chem 60 (1988) 2299, K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, Rapid Commun. Mass Spectrom. 2 (1988) 151-153 and R. C. Beavis and B. T. Chait, Rapid Commun Mass Spectrom 4 (1989) 233 and 432-440, is generally considered to be a soft ionization method with spectra containing mostly molecular ions, but both prompt and metastable fragmentation processes are known to occur. Fragmentation is most readily observed with reflecting analyzers and the technique known as xe2x80x9cpost-source decayxe2x80x9d (PSD) has been developed to provide structural information on peptides and other small molecules (see, e.g., R. Kaufmann, B. Spengler and F. Lutzenkirchen, Rapid Com. M Sp 7(1993) 902-910 (PSD), and R. Kaufmann, P. Chaurand, D. Kirsch, B. Spengler, RCMS, 10 (1996) 1199-1208. Proteins and larger DNA oligomers often fragment extensively in a TOF mass spectrometer between the ion source and the detector, and in some cases the parent ion is poorly detectable in reflecting analyzers. Molecular ions may still dominate the spectra observed in a linear analyzer provided a significant fraction of such ions survives acceleration.
Not all of the ion excitation comes from the desorption process itself. Ions are excited while being dragged through the matrix plume by an accelerating electric field (see R. Kaufmann, P. Chaurand, D. Kirsch, B. Spengler, RCMS, 10 (1996) 1199-1208, R. C. Beavis and B. T. Chait, Chem. Phys. Lett., 181 (1991), 479, A. Verentchikov, W. Ens, J. Martens and K. G. Standing, Proc. 40th ASMS Conf.(1992)p.360, and J. Zhou, W. Ens, K. G. Standing and A. Verentchikov Rapid Commun. Mass Spectrom, 6 (1992) 671). As a result, the best performance, in so far as reduced fragmentation is concerned, is obtained right near the ionization/desorption threshold irradiance , which depends on a particular xe2x80x9csweet spotxe2x80x9d on the matrix crystal.
Delayed ion extraction (DE) (see, e.g., R. S. Brown and J. J. Lennon Anal. Chem 67 (1995, 1998), and M. L. Vestal, P. Juhasz and S. A. Martin, Rapid Commun. Mass Spectrom 9(1995) 1044-1050) partially overcomes the fragmentation problem and makes the MALDI method more robust. In DE, application of the accelerating electric field is delayed so that the plume of neutral molecules desorbed by the laser has expanded sufficiently by the time the field is applied such that collisions are relatively improbable. As a result, stable ions can be obtained over a wider range of laser energy. Introduction of the DE technique strongly improved MALDI performance for peptides, medium mass proteins and DNA. However, performance, even when the DE method is applied, still deteriorates for proteins above 30 kD and mixed DNA larger than 60 mers. The peak shape is affected by unresolved losses of small groups and by adducts. Survival of molecular ion gets worse with size, particularly for DNA molecules. Additionally, the MALDI method is known to form clusters of protein ions with matrix molecules, which deteriorates the resultant spectrum.
The need exists for an improved MALDI source for an MS instrument that is capable of accurately determining the molecular weight of large molecules, particularly proteins and DNA oligomers. It is desirable that such a source achieve accurate molecular weight measurement by producing xe2x80x9ccleanxe2x80x9d spectra through avoiding undue fragmentation and undesirable clustering.
In accordance with various embodiments of the invention, the MALDI technique has been extended for determining the molecular weight of labile molecules, thereby making the technique particularly useful for molecules of biological importance such as peptides, proteins, and DNA oligomers. The invention overcomes the limitations of the prior art with respect to apparatus and methods employing the MALDI technique and thus extends the utility of this technique for labile biopolymers by avoiding uncontrolled fragmentation in some cases, and also undesirable clustering with matrix and impurity molecules. Both of these effects have in the past limited the utility of the MALDI technique for reliably determining molecular weights of biopolymers larger than about 30,000 Da.
The invention is based on the recognition that low energy collisions of excited ions with neutral molecules can cause rapid collisional cooling and thus relax internal excitation and improve the stability of MALDI-produced ions. In accordance with a feature of the invention, recent experimental studies by the inventors have found that losses of small groups and backbone fragmentation are practically eliminated at a MALDI source pressure of around 1 torr. In accordance with another feature of the invention, at room temperature and at gas pressures above 0.1 torr to around 10 torr, the formation of clusters of protein ions with matrix molecules can be efficiently broken without fragmenting proteins by increasing the downstream gas temperature between 150 and 250xc2x0 C. It has also been found desirable to control the temperature in the ion source chamber below 50xc2x0 C. to avoid sample degradation. Stabilization of ions and removal of matrix complexes improves the quality of protein spectra. Isotope limited resolution can be achieved for the 47 kD protein enolase.
Once metastable fragmentation of proteins is prevented by gas collisions, the qualitative aspects of the ionization process in MALDI become highly insensitive to laser fluence. Over a wide range of laser power (almost one decade) spectra remain strikingly similar. In accordance with another feature of the invention, operating at a high laser energy in combination with intermediate gas pressure significantly increases ion signal intensity. Further optimization of ion signal intensity and speed of data acquisition can be achieved by also simultaneously operating the laser at a high repetition rate and controlling the scan rate of the sample plate without saturating the data acquisition system. The invention thus provides for an instrument system that is highly insensitive to variations of laser energy and sample preparation technique.
An objective of this invention is to control and reduce the fragmentation of molecular ions produced by MALDI.
Another objective is to control and reduce the amount of clustering of neutral molecules on molecular ions produced by MALDI.
Other objectives are to improve the sensitivity, resolution, and mass accuracy for molecular weight determinations on large molecules by MALDI-TOF mass spectrometry.
Another objective is to provide apparatus and methods for determining the molecular weight of larger DNA fragments, including mixtures of such fragments which can be used to determine DNA sequence.
These objectives are accomplished by providing apparatus and methods for controlling the pressure and temperature of the neutral gas within the MALDI ion source.
Preferred embodiments are described which are particularly applicable to introduction of ions to a time-of-flight mass spectrometer orthogonally to the direction of ion transport from the source. Other embodiments are described which are also applicable to more conventional xe2x80x9cco-axialxe2x80x9d time-of-flight mass spectrometry in which direction of ion introduction is substantially parallel to the direction of ion motion in the TOF analyzer.